A comparative study of food selectivity of the benthic copepod Tigriopus japonicus and the pelagic copepod Paracyclopina nana was performed by feeding four different microalgae (Tetraselmis suecica, Isochrysis galbana, Chlorella sp., and Nannochloropsis oculata). To examine whether the morphological characteristics of the microalga could influence the food selectivity, we investigated the movement, color, and size of the microalgae. The result indicated significant differences only in the movement of microalga. However, no significant differences in most of the microalgae were observed in feeding rate determination in 24 h, indicating plenty amounts of microalgae were administered to both copepods. In the T. suecica- and I. galbana-fed groups, no significant nitrogen isotopic turnover was observed, indicating both microalgae might be suitable for experimental copepods. However, the difference in the isotopic ratio of N. oculata and N. oculata-fed copepods showed that N. oculata was not a good food source. Various types of polyunsaturated fatty acids were successfully synthesized. However, the class of fatty acid conversion genes was different and the transcript level showed different trends according to each microalgal species in the two copepods. In developmental time, T. japonicus was not severely affected by the food types, while in P. nana, only T. suecica-fed group showed typical development progression. For further verification, both copepods were exposed to frozen-T. suecica. The result showed that T. japonicus was not affected by the frozen-T. suecica-fed condition, but no sign of development in P. nana was shown, suggesting that P. nana could not detect non-motile microalgae.